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Minimal PAM specificity of a highly similar SpCas9 ortholog.

Publication ,  Journal Article
Chatterjee, P; Jakimo, N; Jacobson, JM
Published in: Science advances
October 2018

RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5'-NNG-3' PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.

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Published In

Science advances

DOI

EISSN

2375-2548

ISSN

2375-2548

Publication Date

October 2018

Volume

4

Issue

10

Start / End Page

eaau0766

Related Subject Headings

  • Substrate Specificity
  • Streptococcus
  • Sequence Homology
  • Humans
  • HEK293 Cells
  • Genome, Human
  • Genetic Engineering
  • Gene Editing
  • Computational Biology
  • CRISPR-Cas Systems
 

Citation

APA
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ICMJE
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Chatterjee, P., Jakimo, N., & Jacobson, J. M. (2018). Minimal PAM specificity of a highly similar SpCas9 ortholog. Science Advances, 4(10), eaau0766. https://doi.org/10.1126/sciadv.aau0766
Chatterjee, Pranam, Noah Jakimo, and Joseph M. Jacobson. “Minimal PAM specificity of a highly similar SpCas9 ortholog.Science Advances 4, no. 10 (October 2018): eaau0766. https://doi.org/10.1126/sciadv.aau0766.
Chatterjee P, Jakimo N, Jacobson JM. Minimal PAM specificity of a highly similar SpCas9 ortholog. Science advances. 2018 Oct;4(10):eaau0766.
Chatterjee, Pranam, et al. “Minimal PAM specificity of a highly similar SpCas9 ortholog.Science Advances, vol. 4, no. 10, Oct. 2018, p. eaau0766. Epmc, doi:10.1126/sciadv.aau0766.
Chatterjee P, Jakimo N, Jacobson JM. Minimal PAM specificity of a highly similar SpCas9 ortholog. Science advances. 2018 Oct;4(10):eaau0766.

Published In

Science advances

DOI

EISSN

2375-2548

ISSN

2375-2548

Publication Date

October 2018

Volume

4

Issue

10

Start / End Page

eaau0766

Related Subject Headings

  • Substrate Specificity
  • Streptococcus
  • Sequence Homology
  • Humans
  • HEK293 Cells
  • Genome, Human
  • Genetic Engineering
  • Gene Editing
  • Computational Biology
  • CRISPR-Cas Systems